一、    Principle of test

The bacteriostatic agent is continuously dissolved and diffused by AGAR to form different concentration gradients to show its bacteriostatic effect. The size of the bacteriostatic zone was used to determine whether it had bacteriostatic ability. This test is suitable for the identification of bacteriostatic agents and soluble antifungal products.

二、    Test equipment, chemical reagents and strains

Petri dish, test tube, 5ul~50ul micro pipette, 100ul -~100uL. Trace pipette and supporting gun head, 10mL centrifugal tube, vernier caliper, coating rod, McFarLand turbidimetric tube, electric mixer, water bath, ultra clean table, autoclave, air drying oven, biochemical incubator, electric furnace nutrition AGAR medium, PBS buffer, sterile water, 95% ethyl alcohol.

Strains: Hemolytic Streptococcus, Staphylococcus hemolytic, Listeria, Mycobacterium tuberculosis, coryne bacterium diphtheria, absorbent Pseudomonas aeruginosa, Escherichia coli

三、      Test procedure

3.1 Preparation and sterilization of experimental equipment

The AGAR medium of nutrition is prepared in the conical bottle, heated in the electric furnace and boiled until the state is clarified, the plug is covered, and the PBS buffer, sterile water, supporting gun head, coating rod and centrifugal tube are put into the autoclave for sterilization, and the sterilization is carried out at 121℃ 25mino. The petri dish was wrapped in brown paper and put together with the test tube into a blast oven for sterilization, and sterilization was conducted at 170℃ for 2h. Before the experiment, the ultra-clean working table and operating room should be sterilized with UV lamp for more than 30min before use.

3.2 Pour the petri dish

Place the medium and petri dish to 60℃ and start pouring the petri dish. Shake the medium well before use, pour about 15-20mL into each petri dish, and then stand horizontally until completely solidified.

3.3 Preparation of bacterial suspension

After taking out the strain to be tested from the refrigerator and activating it for 0.5h, add 5mL PBS buffer and gently shake it 80 times on the inclined plane on the palm, so that the strain is completely washed down, then pour it into the test tube and gently shake it well. Absorb 1mL of bacterial solution and add it to 4mL of PBS buffer, and then absorb 1mL of diluted bacterial suspension for 5 times gradient dilution successively, at 10⁸CFU/mL. Around the bacteria Suspension or spore suspension of about 10⁶CFU/mL (compared with 0.5 McFarLand turbidimetric tube), after dilute release of the selected bacterial suspension of 10 times series, select a second test tube (that is, bacterial suspension with a concentration of about 10⁶CFU/mL or spore suspension with a concentration of about 10⁴CFU/mL) for test.

3.4 Inoculate the test bacteria

Use a pipette gun to absorb 0.1mlL of the test bacteria suspension with a concentration of 10cfu/ml, and inoculate it into the nutrient AGAR petri dish which has been inverted. Use a coating stick to coat evenly (pay attention to the coating stick to sterilize under the alcohol lamp after each use, and do not scratch the medium when coating), and cover the petri dish well.

四、    The configuration of bacteriostatic agent

Use an analytical balance to accurately weigh 1g sample in a 15m L centrifuge tube after sterilization, add 10mL solvent (sterile pure water, 95% ethanol, etc.), shake well to dissolve (according to the situation can use an electric mixer to shake well or heat in a water bath to dissolve).

After dissolution, the supernatant is absorbed to double dilute the sample, which can be diluted several times according to the situation, generally diluting 6~10 times. If the sample is liquid, can be directly to double dilution.

五、  Dosage of mildew agent

5.1 Make three holes in the petri dish and punch holes with the gun head of 10 -100uL. After the holes are finished, the culture base in the AGAR holes will be picked out with a sterile needle (drill holes once, do not turn the gun head to prevent cracks in the AGAR ring, which will affect the diffusion of the drug liquid and cause uneven bacteriostatic ring. After each sterile needle is picked, it is necessary to burn the alcohol lamp for sterilization). The center of each hole is more than 25mm apart, and the perimeter of the petri dish is more than 15mm apart. Use a micropipette to suck 20uL of bacteriostasis into the AGAR well (three Wells in a group) and cover the petri dish.

5.2 The petri dish without antibacterial agent and the solvent when adding antibacterial agent were used as positive control, and the uninoculated petri dish was used as negative control. 

5.3 Culture the petri dish and observe the bacteriostatic effect.

It was cultured in a biochemical incubator at 37℃ for 16h~18h to observe the results. The diameter of bacteriostatic zone was measured with vernier caliper and recorded. Note: Do one concentration for each petri dish. When measuring the inhibition zone, choose the one with uniform and completely sterile growth. The diameter should be measured with the outer edge of the inhibition zone as the boundary.

六、   Test results

Diameter of bacteriostatic zone (mm) of each concentration of bacteriostatic agent

Positive control results：     Black     

Negative control result：       0.1% Combat BV    0.2% Combat BV    0.1% Combat MBS 05    0.2% Combat MBS 05 

七、   Evaluation stipulation

7.1 Evaluation of bacteriostasis

Ø  Bacteriostatic zone diameter less than or equal to 7mm, judged as no bacteriostatic effect.

Ø  If the diameter of the bacteriostatic zone is greater than 7mm, it is judged as having bacteriostatic effect.

Ø  If the diameter is greater than 7mm and less than 10mm, it is judged as insensitive; Greater than 10mm and less than 20mm is judged as moderate sensitivity; Greater than 20mm is high sensitivity.

7.2 Three times repeated tests have antibacterial results, judged as qualified.

7.3 Precautions

(1) The concentration of bacterial suspension used for inoculation should meet the requirements. If the concentration is too low, the number of inoculating bacteria is small, and the antibacterial zone is often increased as a result. If the concentration is too high and the inoculation amount is too large, the inhibitory zone can be reduced.

(2) The accuracy of the AGAR concentration should be maintained, otherwise the size of the bacteriostatic zone can be affected.

(3) The culture time should not exceed 18 h. If the culture is too long, part of the bacteria can resume growth, and the antibacterial cost becomes smaller.

(4) The diameter of the bacteriostatic zone can be affected by the am out of bacteriostatic agent, bacteriostatic performance and dry humidity.